Project summary Unraveling complex behavior of healthy and diseased brain by analyzing the structure and dynamics of neural circuitry with single action potential resolution is a long-standing goal for neuroscience. While many voltage-sensitive indicators have been developed for direct imaging of cellular membrane potentials, realization of their in vivo potential is still compromised by toxicity, time resolution and signal weakness arising from nonspecific background labeling, low quantum yields, limited dynamic range and signal dampening from increased cellular capacitance. Here we take a novel approach using de novo designed proteins to secure a transmembrane redox chain of endogenous heme and respond to changes in membrane potential at the speed of electron tunneling. We propose to exploit the adaptability of de novo protein design to gain leveraged microsecond voltage sensitivity, sufficiently fast to resolve the entire action potential waveforms in neurons. Optical detection of the proposed transmembrane electron-transfer relay will be achieved via energy transfer with fused genetically encoded fluorescent proteins. We expect these voltage sensors to be dramatically faster and more tunable than current genetically encoded voltage indicators (GEVIs) based on voltage-dependent protein structural rearrangements with fundamental kinetic limit of ~0.5 millisecond. When developed, these sensors will greatly advance optical imaging of neural activity, thereby accelerating progress toward understanding how brain activity governs human behavior, cognition, and abnormal pathologies.